Nuclear Medicine and Biology 90–91 (2020) 49–54

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Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio[ 131I]MIBG exports via MRP transporters and inhibition of the MRPtransporters improves accumulation of [ 131I]MIBG in neuroblastoma
Masato Kobayashi a,⁎, Asuka Mizutani a, Kodai Nishi b, Yuka Muranaka c, Ryuichi Nishii d, Naoto Shikano e,
Takeo Nakanishi f, Ikumi Tamai g, Eugenie S. Kleinerman h, Keiichi Kawai a,iaSchool of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan
Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japand
Department of Molecular Imaging and Theranostics, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba, Japane
Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japanf
Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japang
School of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, JapanhDivision of Pediatrics, University of Texas M.D. Anderson Cancer Center, Houston, USAi
Biomedical Imaging Research Center, University of Fukui, Fukui, Japanbca r t i c l e

i n f oArticle history:
Received 27 June 2020
Received in revised form 12 September 2020
Accepted 22 September 2020
Keywords:[123/131I]MIBG
Neuroblastoma
Efﬂux transporter
MRP
Probenecida b s t r a c t
Introduction: 131I-labeled m-iodobenzylguanidine ([131I]MIBG) has been used to treat neuroblastoma patients,
but [131I]MIBG may be immediately excreted from the cancer cells by the adenosine triphosphate binding cassettetransporters, similar to anticancer drugs. The purpose of this study was to clarify the efﬂux mechanism of [131I]
MIBG in neuroblastomas and improve accumulation by inhibition of the transporter in neuroblastomas.
Methods: [131I]MIBG was incubated in human embryonic kidney (HEK)293 cells expressing human organic aniontransporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic anion transporter (OAT)1 and OAT2, organic
cation transporter (OCT)1 and OCT2, and sodium taurocholate cotransporting polypeptide, and in vesicles expressing P-glycoprotein (MDR1), multidrug resistance associated protein (MRP)1–4, or breast cancer resistance
protein with and without MK-571 and probenecid (MRP inhibitors). Time activity curves of [ 131I]MIBG withand without MK-571 and probenecid were established using an SK-N-SH neuroblastoma cell line, and transporter
expression of multiple drug resistance was measured. Biodistribution and SPECT imaging examinations were conducted using [123I]MIBG with and without probenecid in SK-N-SH-bearing mice.
Results: [131I]MIBG uptake was signiﬁcantly higher in OAT1, OAT2, OCT1, and OCT2 than in mock cells. Uptake via
OCT1 and OCT2 was little inhibited by MK-571 and probenecid. [ 131I]MIBG uptake into vesicles that highly
expressed MRP1 or MRP4 was signiﬁcantly higher in ATP than in AMP, and these inhibitors restored uptake tolevels similar to that in AMP. Examining the time activity curves for [131I]MIBG in SK-N-SH cells, higher expressions of MDR1, MRP1, MRP4, and MK-571, or probenecid loading produced signiﬁcantly higher uptake than incontrol at most incubation times. The ratios of tumors to blood or muscle in SK-N-SH-bearing mice were significantly increased by probenecid loading in comparison with normal mice.
Conclusions: [131I]MIBG exports via MRP1 and MRP4 in neuroblastoma. The accumulation and tumor-to-blood ormuscle ratios of [131I]MIBG are improved by inhibition of MRPs with probenecid in neuroblastoma.
Advances in knowledge: [131I]MIBG, widely used for treatment of neuroendocrine tumors including neuroblastoma, is excreted via MRP1 and MRP4 in neuroblastoma.
Implications for patient care: Loading with probenecid, OAT, and MRP inhibitors improves [131I]MIBG accumulation.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction
Neuroblastoma, which is derived from the neural crest, is the mostcommon extracranial solid cancer in children [1]. Patients with metasta⁎ Corresponding author at: School of Health Sciences, Institutes of Medical, Pharmaceutical and Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942,
Japan.
E-mail address: kobayasi@mhs.mp.kanazawa-u.ac.jp (M. Kobayashi).
https://doi.org/10.1016/j.nucmedbio.2020.09.004
0969-8051/© 2020 Elsevier Inc. All rights reserved.

tic neuroblastoma usually have a poor prognosis. To improve their poor
prognosis, the accurate identiﬁcation of all lesions is crucial to evaluate
the extent of the neuroblastoma [2].
Iodine-123 labeled m-iodobenzylguanidine ([ 123I]MIBG) and [ 131I]
MIBG, an analog of the adrenergic neurotransmitter norepinephrine,
have been used as adrenomedullary scintigraphic agents with their
gamma-rays since the early 1980s [3–5] and for detecting neuroendocrine tumors such as neuroblastoma, pheochromocytoma, and medullary thyroid cancer [6]. At present, [ 123I]MIBG is routinely used forM. Kobayashi, A. Mizutani, K. Nishi et al.

Nuclear Medicine and Biology 90–91 (2020) 49–54

staging and follow-up of patients with neuroendocrine tumors and issuitable for [ 131I]MIBG therapy. Because it emits beta rays with cytocidal
effects, [ 131I]MIBG has been used for the treatment of neuroendocrine
tumors including neuroblastoma as a primary agent and in combination
with other therapies [7,8].
In human cancers, organic anion transporting polypeptide (OATP)
1B1 (SLCO1B1), OATP1B3 (SLCO1B3), OATP2B1 (SLCO2B1), organic
anion transporter (OAT)1 (SLC22A6), OAT2 (SLC22A7), organic cationtransporter (OCT)1 (SLC22A1), OCT2 (SLC22A2), Na +-taurocholate
cotransporting polypeptide (NTCP) (SLC10A1) isoforms are expressed
as solute carrier (SLC) transporters [9,10]. Many anticancer drugs andmedicines are incorporated into tumors via these SLC transporters forcancer treatment.
When multiple drug resistance (MDR) with ATP-binding cassette(ABC) transporters occurs in cancer cells, anticancer drugs accumulate
in the cancer cells and are then immediately excreted from the cancer
cells [11,12]. Thus, the total amount of the anticancer drug decreases
in the cancer cells, and the therapeutic effect decreases. The ABC transporters are classiﬁed into seven subfamilies based on phylogenetic analysis. In tumor cells, ABC proteins mainly include P-glycoprotein (P-gp
and MDR1, gene symbol ABCB1), multidrug resistance protein 1(MRP1, gene symbol ABCC1), MRP2 (gene symbol ABCC2), MRP3 (genesymbol ABCC3), MRP4 (gene symbol ABCC4), and breast cancer resistance protein (BCRP, gene symbol ABCG2).
Since the effect of internal radiotherapy with [ 131I]MIBG is not thesame in all patients, there is uptake of [ 131I]MIBG into the cancer cells
once, and it may be immediately excreted from the cancer cells by the
ABC transporters, as in the case of anticancer drugs. Thus, the therapeutic
effect of [ 131I]MIBG may change in every patient based on the total
amount of [131I]MIBG accumulated in tumor cells. If the injected radioactivity of [131I]MIBG was increased, uptake of [ 131I]MIBG into neuroblastoma may be improved, but whole-body radiation exposure would behigher. An alternative strategy is needed to improve [131I]MIBG accumulation in tumor cells.
Although it has been reported that P-gp is not involved in efﬂux of[131I]MIBG [13], the relationship between [131I]MIBG and other ABC transporters has not been evaluated. The purpose of this study was to elucidate
the efﬂux mechanism of [131I]MIBG in neuroblastomas and improve accumulation by inhibition of the transporters in neuroblastomas.

As ABC transporters, vesicles (GenoMembrane Inc., Kanagawa,Japan) with high expression of human MDR1, MRP1–4, and BCRP
were used. Experimental kits were also purchased from GenoMenbrane
Inc. and used for experiments with each ABC transporter.
2.3. Uptake experiments with HEK293 cells
First, expression levels of SLC transporters were conﬁrmed in the
HEK293 cells expressing OATP using [6,7- 3H(N)]estrone-3-sulfate(PerkinElmer Inc., Waltham, MA, USA, 2.04 GBq/mmol, 2.22 TBq/mmol),OAT using p-[14C]aminohippuric acid (PerkinElmer Inc., 2.04 GBq/mmol), OCT using [ 3H]methyl-4-phenylpyridinium (American
Radiolabeled Chemicals Inc., St. Louis, MO, USA, 2.96 TBq/mmol), and
NTCP using [ 3H(G)]taurocholic acid (PerkinElmer Inc., 37 GBq/mmol).
One day before the uptake experiments, HEK293 cells expressing an SLC
transporter were prepared at 4 × 105 cells/well in 12-well plastic plates.
Cells were pre-incubated for 10 min using modiﬁed Hank's balanced
salt solution. Each cell was incubated with [ 131I]MIBG (37 kBq) for
5 min (n = 4) and removed from the tissue culture by 0.25% trypsinEDTA solution (Sigma-Aldrich). Then, the radioactivity of the cells wasmeasured using a gamma counter. For protein assays of the cells, cellular
protein content was measured with a BCA protein assay kit (Thermo
Fisher Scientiﬁc Inc., Waltham, MA, USA) using BSA as a standard. Uptakesof [131I]MIBG in HEK293/OATP1B1, HEK293/OATP1B3, HEK293/OATP2B1,
HEK293/OAT1, HEK293/OAT2, HEK293/OCT1, HEK293/OCT2, andHEK293/NTCP were compared with that in mock HEK293 cells. Using assays with inhibitors, uptake of [ 131I]MIBG was examined in HEK293/
OAT1, HEK293/OAT2, HEK293/OCT1, and HEK293/OCT2 cells with MK571, a substrate for OATs [15] and an MRP inhibitor [16,17], and probenecid, a substrate for OATs [18], OCTs [19], and MRP inhibitors [16,17]. The
uptake of [131I]MIBG is shown as % injected dose (%ID)/mg protein.
2.4. Uptake experiments with vesicles
After pre-incubation of vesicles for 10 min using reaction buffer including 50 mM MOPS-Tris, 70 mM KCl, and 7.5 mM MgCl2 in the kit,
37 kBq [ 131I]MIBG were incubated for 5 min with each vesicle solution
and ATP (n = 4), which supplies energy for ABC transporters, or adenosine monophosphate (AMP, n = 4), which does not provide energy
and was used for comparison to ATP, on nitrocellulose ﬁlters, and radioactivity was measured using a γ-ray counter (AccuFLEXγ7000, Aloka,
Tokyo, Japan). Uptake of [ 131I]MIBG in ATP solution was comparedwith that in AMP solution. When uptake of [ 131I]MIBG in ATP solution
was higher than that in AMP solution, this indicated an effect of 131IMIBG on ABC transporters. In assays with inhibitors, uptake of [ 131I]
MIBG was examined in ATP solution with MK-571 and probenecid(MRP inhibitors) using vesicle solution with high expressions of MRP1
and MRP4. The uptake of [ 131I]MIBG is shown as nmol/mg protein.

2. Material and methods
2.1. Materials[123I]MIBG (74 MBq/mL, more than 95% purity) for biological distribution and single photon emission computed tomography (SPECT) imaging,
instead of [131I]MIBG and [131I]MIBG (370 MBq/mL), was purchased from
FUJIFILM Toyama Chemical Co., Ltd. (Tokyo, Japan) and IZOTOP Instituteof Isotopes Co., Ltd. (Budapest, Hungary), respectively. MK-571 sodium
salt and probenecid were purchased from Cayman Chemical (AnnArbor, MI, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.

2.5. SK-N-SH neuroblastoma cell line
The cultured human cancer cell line SK-N-SH neuroblastoma waspurchased from American Type Culture Collection (Manassas, VA,
USA). Cancer cells were incubated in αMEM (Wako, Osaka, Japan)
with 10% fetal bovine serum.

2.2. Human embryonic kidney (HEK)293 cells for SLC transporters and vesicles for ABC transporters
As SLC transporters, HEK293 cells expressing OATP1B1, OATP1B3,
OATP2B1, OAT1, OAT2, OCT1, OCT2, and NTCP plasmid vector alone formock cells were prepared as described previously [14]. Brieﬂy, HEK293
cells were transfected with the respective plasmid DNA, and they werethen selected with the appropriate antibiotics; HEK293/OATP1B1,
HEK293/OATP1B3, HEK293/OATP2B1, HEK293/OAT1, HEK293/OAT2,HEK293/OCT1 HEK293/OCT2, and HEK293/NTCP cells and mock cells
were designed. All cell lines were grown in Dulbecco's modiﬁed eagle medium (Wako Pure Chemical Industries Ltd., Osaka, Japan) supplemented
with 10% (v/v) fetal bovine serum (Life Technologies, Carlsbad, CA),
100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C and 5% CO2.

2.6. Expression of ABC transporters in SK-N-SH cells using real-time PCR
Expression of ABC transporters in human cancer cells was evaluatedas described [19]. The following genes were analyzed using real-timepolymerase chain reaction with an Mx3005P thermocycler (Agilent
Technologies, Santa Clara, CA, USA): MDR1 (ABCB1) and MRP1, 2, 3,
and 4 (ABCC1, 2, 3 and 4) and BCRP (ABCG2). Three different housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta
actin (ACTB), and hypoxanthine phosphoribosyltransferase-1 (HPRT1),
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Nuclear Medicine and Biology 90–91 (2020) 49–54

Fig. 1. Uptake of [131I]MIBG by HEK293 cells expressing an SLC transporter (n = 4). [131I]MIBG uptake is signiﬁcantly higher in HEK293/OAT1, HEK293/OAT2, HEK293/OCT1, and HEK293/
OCT2 cells than in mock cells. Although the uptake of HEK293/OAT1 and HEK293/OAT2 cells is signiﬁcantly inhibited by MK-571 and probenecid, the uptake of HEK293/OCT1 and HEK293/
OCT2 cells is little inhibited. *P < 0.05 vs. mock cells, between control of HEK293 cells and HEK293 with MK-571 or probenecid loading.

Scid mice (female, 5 weeks old, SLC Inc., Hamamatsu, Japan) weretransplanted with SK-N-SH cells (5 × 10 5 cells/100 μL) and Matrigel(#354230, Corning, Corning, NY, USA) into the lower abdomen of themice. The mice were housed for about 5–7 weeks under a 12-h light/12h dark cycle with free access to food and water. The mice were fasted
with no food overnight with water supplied ad libitum before experiments. About 1 month later, the size of the SK-N-SH tumor became palpable with a diameter of 5–10 mm. Then, 200 kBq [123I]MIBG were injected
via the tail vein in SK-N-SH-bearing mice. Mice were sacriﬁced at 10, 30,
60, and 120 min post-injection (each n = 3). After blood was sampled
via cardiocentesis, brain, heart, liver, kidney, muscle, and the SK-N-SH
tumor were excised. Radioactivity in weighed tissue samples was measured using a gamma counter (ARC-380; Aloka). Data are expressed as
injected dose per g wet tissue (%ID/g tissue). For the loading study of probenecid, which has a high safety level for humans, the SK-N-SH-bearing
mice were injected with a mixture of [123I]MIBG and 1 mM probenecid,
and the distribution studies were performed using the same protocolthat was used for SK-N-SH-bearing mice without probenecid loading as
control mice.

were ampliﬁed to control for the differences between the initial RNA
and cDNA amounts.
2.7. Transport assays with SK-N-SH
Transport assays were performed as described [20]. One day after
seeding of SK-N-SH cells, each well was pre-incubated with 1 mL of incubation medium for 10 min. The cells were then incubated with 37 kBq[131I]MIBG for 1, 3, 5, 10, 30, 60, 120, or 180 min at 37 °C (n = 4). For
the competitive inhibition assay, the cells were incubated for the above incubation time with [131I]MIBG in the presence of inhibitor: ﬁnal concentration 50 μM MK-571 sodium salt [16,17] or 1 mM probenecid for MRP1–4[16,17]. At the end of the incubation, each well was rapidly washed twicewith 1 mL of ice-cold incubation medium. The cells were then solubilized
in 0.5 mL 0.1 N NaOH, and radioactivity was measured with a γ-ray counter. The uptake of [131I]MIBG is shown as nmol/mg protein.
2.8. Biological distribution of [123I]MIBG in SK-N-SH-bearing mice
All applicable institutional guidelines for the care and use of animals
were followed at Kanazawa University. All procedures performed in studies involving animals were in accordance with the ethical standards of Kanazawa University (the Animal Care Committee of Kanazawa University,
AP-122339) and were conducted in accordance with the international
standards for animal welfare and institutional guidelines. Twenty-four

2.9. SPECT imaging with [123I]MIBG in SK-N-SH-bearing mice
[123I]MIBG (30–40 MBq, n = 3) or a mixture of [123I]MIBG and 1 mM
probenecid (n = 3) were injected into the tail vein of SK-N-SH-bearing
mice, SPECT acquisition was started 5 min after injection, and SPECT im-

Fig. 2. Uptake of [131I]MIBG by vesicles overexpressing each ABC transporter (n = 4). [131I]MIBG uptake into vesicles that highly expressed MRP1 or MRP4 in the presence of ATP issigniﬁcantly higher than that in the presence of AMP, but not MDR1, MRP2, MRP3, and BCRP. Uptake via MRP4 is higher than that via MRP1. In MK-571 or probenecid loading, uptakeof [131I]MIBG decreases to levels similar to that in AMP solution. *P < 0.05 vs. presence of AMP, ATP with MK-571 or probenecid loading.
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Nuclear Medicine and Biology 90–91 (2020) 49–54

Data are presented as means and standard deviation (SD). P values
were calculated using the two-tailed paired Student's t-test for comparison between two groups or analysis of variance and Dunnett's test,
which were used as a multiplex analysis with a population mean value,
using GraphPad Prism 7 statistical software (GraphPad Software, Inc., La
Jolla, CA, USA). A P value less than 0.01 or 0.05 was considered signiﬁcant.

HEK293/OAT1, HEK293/OAT2, HEK293/OCT1, and HEK293/OCT2 cells
than in mock cells. Although the uptake of HEK293/OAT1 andHEK293/OAT2 cells was signiﬁcantly inhibited by MK-571 and probenecid, the uptake of [131I]MIBG via HEK293/OCT1 and HEK293/OCT2 cells
was little inhibited. Fig. 2 shows the uptake of [ 131I]MIBG by vesicles
overexpressing each ABC transporter. [ 131I]MIBG uptake into vesicles
that highly expressed MRP1 or MRP4 in the presence of ATP was significantly higher than that in the presence of AMP, but not MDR1, MRP2,
MRP3, and BCRP. MK-571 or probenecid loading in vesicle solution
with high expression of MRP1 or MRP4 in the presence of ATP solution
restored uptake levels of [ 131I]MIBG similar to that in AMP solution.
After conﬁrming the high quality of the total RNA harvested from SKN-SH cells using a bioanalyzer (data not shown), genes for transportersof MDR1 (25.83 as the copy number per 1000 copies of housekeeping
genes), MRP1 (23.41), and MRP4 (6.76) were highly expressed in comparison with MRP2 (0.16), MRP3 (0.75), and BCRP (0.00). In the time activity curves of [ 131I]MIBG in SK-N-SH cells (Fig. 3), MK-571 orprobenecid loading produced signiﬁcantly higher uptake than in thecontrol at most incubation times. At 120 min of incubation, uptake of[ 131]MIBG with MK-571 loading was about 1.4-fold higher than in thecontrol, and that with probenecid loading was about 1.5-fold higher.
However, there was no signiﬁcant difference between MK-571 loadingand probenecid loading.
The biological distribution (Table 1) and SPECT images (Fig. 4) of SKN-SH-bearing mice injected using [ 123I]MIBG with or without probenecid loading were obtained. With respect to the biological distribution of[ 123I]MIBG, [ 123I]MIBG accumulated mainly in blood, heart, liver, kidney,
and SK-N-SH tumors. [ 123I]MIBG injection with probenecid loading resulted in high accumulation in heart, liver, kidney, and SK-N-SH tumors
at 30 min and/or 60 min after administration in comparison with normal mice. At 120 min after administration, accumulation in the heart
and SK-N-SH tumors with probenecid loading increased signiﬁcantly
compared with normal mice. On SPECT images at 10–15 min after[ 123I]MIBG with and without probenecid loading, radioactivity (%ID/g
mice weight) of [ 123I]MIBG was 4.8 ± 3.1 and 3.8 ± 2.9 in heart,
5.1 ± 1.7 and 3.3 ± 1.8 in liver, 3.0 ± 1.3 and 2.6 ± 1.1 in kidney and1.5 ± 0.5 and 1.0 ± 0.4 in SK-N-SH tumors with and without probenecidloading, respectively. At 60–65 min after [ 123I]MIBG with and without
probenecid loading, 4.9 ± 0.8 and 3.4 ± 0.8 in heart, 5.7 ± 1.2 and
4.3 ± 1.1 in liver, 1.7 ± 0.5 and 1.2 ± 0.3 in kidney and 1.4 ± 0.4 and
0.8 ± 0.2 in SK-N-SH tumors, respectively.

3. Results

4. Discussion

Fig. 1 shows uptake of [ 131I]MIBG by HEK293 cells expressing an SLC
transporter. [ 131I]MIBG uptake was signiﬁcantly higher in control ofIn this study, the efﬂux mechanism of [131I]MIBG was clariﬁed, and theaccumulation of [131I]MIBG in neuroblastoma was improved. The inﬂux

Fig. 3. Time activity curves for [131I]MIBG in SK-N-SH cells (n = 4). MK-571 or probenecidloading produces signiﬁcantly higher uptake than in normal mice at most incubation
times. At 120 min of incubation, uptake of [131]MIBG and MK-571 loading is about 1.4fold higher than in the control, and that with probenecid loading is about 1.5-foldhigher. However, there is no signiﬁcant difference between MK-571 loading andprobenecid loading. * P < 0.05 vs. control.

ages were obtained at 10–15 min and 60–65 min using a U-SPECT-II/CT
system (MILabs, Utrecht, The Netherlands). The data were reconstructed using the ordered subset expectation maximization methodwith 16 subsets and six iterations, including no scatter and attenuation
correction. The voxel size was set to 0.8 × 0.8 × 0.8 mm 3. Postreconstruction smoothing ﬁltering was applied using a 1.0-mm Gaussian ﬁlter. Image displays were obtained and analyzed using medical
image data analysis software, AMIDE (ver. 1.0.4). Coronal images are
displayed as similar section images.
2.10. Statistical analysis

Table 1
Biological distribution of [123I]MIBG in mice with and without probenecid loading.

Normal mice

Probenecid loading

Organ (%ID/g)

10 min30 min60 min

120 min

Blood
Brain
Heart
Liver
KidneyMuscle
Tumor
Blood
Brain
Heart
Liver
KidneyMuscleTumor

1.90 ± 0.45
0.30 ± 0.06
28.98 ± 1.72
12.85 ± 3.18
9.13 ± 1.85
1.82 ± 0.49
3.12 ± 0.83
1.98 ± 0.32
0.20 ± 0.05
29.65 ± 0.91
13.12 ± 4.02
10.21 ± 1.67
1.80 ± 0.26
3.34 ± 0.83

1.66 ± 0.65
0.20 ± 0.08
23.14 ± 2.03
23.97 ± 4.53
8.07 ± 1.69
1.55 ± 0.48
2.53 ± 0.72
1.70 ± 0.59
0.15 ± 0.06
25.82 ± 0.93⁎
28.31 ± 6.83⁎
9.52 ± 1.44⁎

1.42 ± 0.12
0.19 ± 0.08
19.47 ± 1.95
18.65 ± 3.83
6.42 ± 1.73
1.48 ± 0.42
2.01 ± 0.63
1.43 ± 0.18
0.14 ± 0.08
21.02 ± 1.05⁎
20.13 ± 4.58⁎
7.82 ± 1.45⁎

1.58 ± 0.32
2.78 ± 0.81

1.35 ± 0.26
2.38 ± 0.71⁎

1.33 ± 0.11
0.17 ± 0.07
16.65 ± 1.89
16.11 ± 3.46
3.89 ± 1.59
1.38 ± 0.39
1.84 ± 0.60
1.34 ± 0.13
0.14 ± 0.06
18.94 ± 1.32⁎
17.12 ± 2.74
3.94 ± 1.59
1.28 ± 0.36
2.17 ± 0.69⁎%ID/g indicates percent injected dose per gram of tissue. Values are the means ± standard deviation obtained from three mice.
⁎ P < 0.05 compared with normal mice.
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Fig. 4. Representative [123I]MIBG SPECT imaging without (a, c) and with probenecid loading (b, d) at 10–15 min (a, b) and 60–65 min (c, d) after administration.

mechanism of [131I]MIBG was investigated to improve the accumulationof [ 131I]MIBG using inhibitors of efﬂux transporters in neuroblastoma.
Bayer has reported that [ 131I]MIBG uptake into neuroblastoma occurs
via OCT3, for which another name is norepinephrine transporter, [20],and the present results showed that [ 131I]MIBG uptake involves OCT1
and OCT2 (Table 1). Anion drugs are usually transported by OATs, which
are SLC transporters, and MRPs in the cells [21]. Although uptake of[131I]MIBG was inhibited by MK-571 and probenecid in HEK293/OAT1
and HEKOAT2 cells (Fig. 1) because MK-571 [15] and probenecid[18,22] are mainly transported by OATs, uptake of [131I]MIBG was little
inhibited by MK-571 and probenecid in HEK293/OCT1 and HEK293/
OCT2 cells (Fig. 1). In assays with vesicles (Fig. 2), [131I]MIBG uptake
into vesicles with high expression of MRP1 or MRP4 was signiﬁcantly
higher in ATP solution than in AMP solution, but not with MDR1, MRP2,
MRP3, and BCRP. Therefore, MK-571 or probenecid was loaded in MRP1
or MRP4 in ATP solution, and similar uptake levels of [131I]MIBG to thatin AMP solution were obtained. In the SK-N-SH cells (Fig. 3) and examining gene expression in SK-N-SH cells, [131I]MIBG efﬂuxed via MRP1 and
MRP4 in neuroblastomas. [131]MIBG with MK-571 or probenecid loading
was given to SK-N-SH cells, efﬂux of [131I]MIBG was inhibited by MK-571
or probenecid, and then the accumulation of [131I]MIBG was signiﬁcantly
increased in SK-N-SH cells (Fig. 3). The increase rate was slightly higher
with probenecid than with MK-571. Since probenecid has higher safety
and easier clinical application for human use than MK-571 [16,23], improvement of the effect of internal radiation therapy in neuroblastoma
using co-injection of [131I]MIBG and probenecid was evaluated.
With respect to the biological distribution in SK-N-SH-bearing micewith probenecid loading (Table 1), [ 123I]MIBG with probenecid loading
provided signiﬁcantly higher accumulation in heart, liver, kidney, and
SK-N-SH tumors at 30 min and/or 60 min after administration compared with in normal mice. Since MRP1 and/or MRP4, which are involved in efﬂux of [ 131I]MIBG, are expressed in heart, liver, and kidney[12] and the SK-N-SH tumors that were evaluated, the accumulationof [123I]MIBG was increased 1.18 and 1.18 times at 60 min and 120 min,respectively, after [ 123I]MIBG administration with probenecid loading
through inhibition of [ 123I]MIBG efﬂux via MRP1 and/or MRP4. It was
found that, 120 min after administration of [123I]MIBG with probenecidloading, washout of [ 123I]MIBG in heart, liver, kidney and SK-N-SH tumors was slower than in normal mice. The effect of probenecid loading
may be maintained due to the high biostability of [123/131I]MIBG [24,25]
and probenecid [16]. Therefore, slower washout of [ 131I]MIBG in heart,
liver and kidney may yield increase of radiation dose by the probenecidloading. The ratios of SK-N-SH tumors to blood and of SK-N-SH tumors
to muscle were evaluated to examine image contrast in SPECT imaging.
The ratio of SK-N-SH tumors to blood was signiﬁcantly increased by probenecid loading in comparison with normal mice at 30 min, 60 min, and
120 min after administration, while the ratio of SK-N-SH tumors to muscle was signiﬁcantly increased by probenecid loading in comparison
with normal mice at all injection times (Table 2). The higher ratios of
SK-N-SH tumors to blood or SK-N-SH tumors to muscle show higher
contrast images between SK-N-SH tumors and backgrounds. On SPECT
imaging using [ 123I]MIBG with probenecid loading at 60–65 min afteradministration, accumulation of [ 123I]MIBG was increased in heart,
liver, kidney, and SK-N-SH tumors compared with normal mice (Fig. 4).
The potential to improve internal radiation therapy with [131I]MIBG
for neuroblastoma using a combination of [131I]MIBG and MRP inhibitors
should be demonstrated. Although probenecid shows high safety, itsometimes has common side effects, such as an allergic reaction, withhigh-dose loading. Additional experiments to determine Kaplan-Meiersurvival estimates, etc. with neuroblastoma-bearing mice are needed toevaluate improvement of internal radiation therapy with [131I]MIBG.
5. Conclusion[ 131I]MIBG is exported via MRP1 and MRP4 in neuroblastoma. The
accumulation and tumor-to-blood or muscle ratios of [ 131I]MIBG

Table 2
Ratios of SK-N-SH tumors to blood and SK-N-SH tumors to muscle.

Normal mice
Probenecid loading

Tumor to blood ratio
Tumor to muscle ratio
Tumor to blood ratio
Tumor to muscle ratio

10 min30 min60 min

120 min

1.64 ± 0.51
1.71 ± 0.45
1.69 ± 0.55
1.86 ± 0.49⁎

1.52 ± 0.71
1.63 ± 0.55
1.64 ± 0.61⁎
1.76 ± 0.58⁎

1.42 ± 0.61
1.36 ± 0.45
1.66 ± 0.58⁎
1.76 ± 0.50⁎

1.38 ± 0.40
1.33 ± 0.42
1.62 ± 0.51⁎
1.70 ± 0.48⁎⁎ P < 0.05 compared with normal mice.
53

M. Kobayashi, A. Mizutani, K. Nishi et al.

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improves with probenecid by inhibition of these transporters inneuroblastoma.
Financial support
This study was funded in part by Grants-in-Aid for Scientiﬁc Research from the Japan Society for the Promotion of Science (Nos.
15K09949, 15K15452, 16KK0200, 16H05397).
Author contributions
Masato Kobayashi was the research coordinator for the study.
Asuka Mizutani and Kodai Nishi performed biodistribution and
SPECT imaging.
Ryuichi Nishi and Naoto Shikano were advisers for transport assays.
Takeo Nakanishi and Ikumi Tamai were advisors for vesicle assays.
Eugenie S. Kleinerman was an advisor for handling of neuroblastoma.
Keiichi Kawai was an advisor for handling of radiopharmaceuticals.
Acknowledgments
The authors would like to thank Mikie Ohtake and other staff of the
School of Health Sciences, Kanazawa University.
Declaration of competing interest
There is no conﬂict of interest.
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